This invention relates to a monoclonal antibody having high specificity against human manganese-superoxide dismutase (hereinafter abbreviated as human Mn-SOD), a method for producing the monoclonal antibody, an assay reagent or assay kit and assay method by use thereof, and a diagnostic method of human ovarian cancer and myocardial infarction by use thereof.
Human Mn-SOD is an enzyme (with a molecular weight of one domain being about 25,000, and comprising a dimer or tetramer thereof) existing in the matrix portion of mitochondrion, and catalyses the reaction of disproportionating superoxide anion radical (O.sub.2.sup.-) which is the main molecular species of active oxygen as shown below: EQU 2 O.sub.2.sup.- +2 H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2.
Whereas, female gynecological cancers include cervical cancer of the uterus, endometrial cancer of the uterus, ovarian cancer, choriocarcinoma, etc. Among them, generation of cervical cancer of the uterus has been constantly reduced year by year, and the choriocarcinoma can be now expected to have high therapeutical effect. However, endometrial cancer of the uterus and ovarian cancer have many problems in an early diagnostic method, therapeutical method, monitoring of prognosis, etc.
Among them, ovarian cancer is generated in all the classes of age from the classes of infant and young women to old women, and is increasing year by year, particularly at a greater ratio in women of fifties or older [Mori, Miyake: Japanese Journal of Cancer Research (Gan), vol. 79, No. 12, 1988]. Also, in therapy of a malignant group of ovarian cancers with extremely poor prognosis, it has been earnestly desired to establish an excellent early diagnosis method.
Accordingly, as the method for diagnosis of ovarian cancer up to date, there are examples in which .alpha.-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin or CA125 which is the cell surface sugar chain antigen of cultured cell line derived from ovarian cancer, etc. are used as the diagnosis marker for investigation (Masato Mochizuki: Rinsho Byori (Clinical Pathology), vol. 34, No. 11, 1986; Kazuo Omi et al: The 9th Clinical Chemical Test Technology Course Text, 1989). However, in the above method, in the case of .alpha.-fetoprotein, probably because it was developed as the diagnosis marker for hepatoma, the positive ratio for epithelial ovarian cancer is 0%; in the case of carcinoembryonic antigen, the positive ratio for epithelial ovarian cancer is 12%; and in the case of human chorionic gonadotropin, the positive ratio for epithelial ovarian cancer is 0%. On the other hand, in the case of CA125 which is the cell surface sugar chain antigen of cultured cell line derived from ovarian cancer, the positive ratio for epithelial ovarian cancer is high as 94%, but exhibits 21% for endometrial cancer of the uterus, and also 30% for endometriosis. Therefore, for diagnosis of epithelial ovarian cancer by use of these diagnosis markers, all of the methods involve problems in their positive ratios and/or specificities.
On the other hand, myocardial infarction is a disease causing ischemia, myocardiopathy, necrosis of myocardium by occlusion and stenosis through the coronary artery, and the acute stage lethality is still at a high value, thus posing a social problem also because of sudden sideration in the generation of people in their prime.
Accordingly, as a countermeasure for its therapy, it is necessary to know the site of myocardial infarction, the size, the course and the prognosis of the lesion by changes in a electrocardiogram, left ventriculography contrast, and fluctuation in serum enzyme.
Fluctuation in serum enzyme is the biochemical diagnostic method to know the fluctuation in deviated enzyme freed out because of myocardial necrosis, and as such method, there have been known the methods in which GOT (glutamic oxaloacetic transaminase), CPK (creatin phosphokinase), LDH (lactic dehydrogenase), etc. are used.
However, according to the biochemical diagnostic methods by use of these enzymes, the enzyme amount exhibits the maximum value within a short time from the onset of stroke of myocardial infarction in most cases, and since these peaks of enzyme coronary angioplasty (the catheter method) (Yoichi Shimizu et al; Nippon Rinsho (Japanese Clinic), vol. 45, 1987, extra No.), percutaneous transluminal coronary recanalization (Katsuo Uematsuse et al: Nippon Rinsho (Japanese Clinic), vol. 45, extra No., 1987), etc. which are the principal methods of myocardial infarction, it is difficult to diagnose accurately myocardial infarction, and also there is the problem that frequent blood sampling is required at the initial stage from a stroke patient because these enzymes have a short time for persistence of a high value of concentration in blood.
On the other hand, it has been considered that a high diagnostic significance exists in the assay of human Mn-SOD concentration in serum in liver disease by the investigations by use of a polyclonal antibody (Iyaku Journal (Journal of Pharmaceutical): Inagaki, Sawaki, 20, 1, 1984; The 5th Marker Research Society, Iizuka, Arai et al, 1985). Also, Taniguchi et al reported that human Mn-SOD concentration was high in lung cancer tissue by the immunological method by use of a polyclonal antibody prepared by immunization of goat (Journal of National Cancer Institute, 72, 5, 1984), and importance of an assay of human Mn-SOD concentration in serum is pointed out.
However, since polyclonal antibodies are used in an assay of human Mn-SOD concentration in serum in these various diseases, it is necessary to produce a monoclonal antibody and establish a method for production thereof in order to further enhance the specificity of the reaction for human Mn-SOD and also obtain constantly an antibody having a high specific reactivity of the same quality, but there has been yet recognized no report concerning a monoclonal antibody having a very high specific reactivity against human Mn-SOD.
A first object of the present invention is to provide a monoclonal antibody having a very high specific reactivity against human Mn-SOD in human serum, and a method for producing the monoclonal antibody.
A second object of the present invention is to provide an assay reagent or an assay kit and an assay method of human Mn-SOD by use of a monoclonal antibody having a very high specific immunoreactivity against human Mn-SOD.
A third object of the present invention is to provide a novel diagnostic method of human epithelial ovarian cancer such as epithelial carcinogenesis of the human ovary and monitoring of its prognosis by assaying the human Mn-SOD concentration in human body fluid by use of the assay reagent or the assay kit of human Mn-SOD comprising a monoclonal antibody as mentioned above.
A fourth object of the present invention is to provide a novel method of myocardial infarction such as sideration of myocardial infarction and monitoring of its prognosis by assaying the human Mn-SOD in human body fluid by use of the assay reagent or the assay kit of human Mn-SOD comprising a monoclonal antibody as mentioned above.
The present inventors have studied intensively in order to solve the above problems and consequently found that a monoclonal antibody exhibiting a very high specific reactivity against human Mn-SOD can be produced by culturing the cell line obtained by immunizing a mouse with human Mn-SOD followed by cell fusion; that human Mn-SOD can be assayed easily and at a high sensitivity by assaying human Mn-SOD by use of an assay reagent or an assay kit of human Mn-SOD by use of the above antibody; that diagnosis of ovarian cancer such as epithelial carcinogenesis of the human ovary and monitoring of its prognosis can be easily performed at a high positive ratio and specificity by assaying human Mn-SOD concentration in human body fluid by use of the assay reagent or the assay kit; and that the diagnosis method of myocardial infarction by use of the assay reagent or the assay kit of human Mn-SOD is not affected at all by reperfusion after therapy, and also requires no frequent blood sampling, and therefore diagnosis such as sideration of myocardial infarction and monitoring of its prognosis can be easily performed, to accomplish the present invention.